Variable phase contrast microscopy

ABSTRACT

In phase contrast microscopy, the phase plate is broken down into two zones, the conjugate and complementary zones, the light beam is polarized in two directions inclined relatively at 45*. One polarization affects the conjugate zone; the other polarization affects the complementary zone, without optical path delay. The apparatus employs a polarizer, a phase plate, a birefringent compensator, and an analyzer having a phase plate with two polarizers turned 45* and respectively occupying the conjugate zone and the complementary zone of the plate. One polarizer is positioned in front of the plate so it covers the two zones and is parallel to the polarizer at the conjugate zone. The birefringent compensator and analyzer are mounted behind the plate, in the direction of light propagation, so that the transmissions of complex amplitudes of the conjugate zone will be 1-ei and that of the complementary zone will be 1.

United States Patent OTHER REFERENCES Jerrard,- Optical Compensators"J.O.S.A. Vol. 38, No. 1, (Jan, 1948) pp. 35- 59.

Primary Examiner-David Schonberg Assistant Examiner-Paul R. MillerAttorney-Littlepage, Quantance, Wray & Aisenberg ABSTRACT: In phasecontrast microscopy, the phase plate is broken down into two zones, theconjugate and complementary zones, the light beam is polarized in twodirections inclined relatively at 45. One polarization affects theconjugate zone; the other polarization affects the complementary zone,without optical path delay. The apparatus employs a polarizer, a phaseplate, a birefringent compensator, and an analyzer having a phase platewith two polarizers turned 45 and respectively occupying the conjugatezone and the complementary zone of the plate. One polarizer ispositioned in front of the plate so it covers the two zones and isparallel to the polarizer at the conjugate zone. The birefringentcompensator and analyzer are mounted behind the plate, in the directionof light propagation, so that the transmissions of complex amplitudes ofthe conjugate zone will be 1 e and that of the complementary zone willbe 1.

[72] lnventor Georges Nomarski Bourg la Reine, France [21] Appl. No.887,621 [22] Filed Dec. 23, 1969 [45] Patented Dec. 21,1971 [73]Assignee Etahlissement Public: Agence Nationale de Valorisation de laRecherche "ANVAR" Hauts-de-Seine, France [54] VARIABLE PHASE CONTRASTMICROSCOPY 8 Claims, 8 Drawing Figs.

[52] U.S.Cl 350/13, 350/12, 350/14. 350/157 [51] Int.C1 ..G02b 27/28[50] Field otSearch 350/12-15, 157; 505/A [56] References Cited UNITEDSTATES PATENTS 2,516,905 8/1950 Osterberg et al. 350/13 2,700,918 2/1955Osterberg et al. 350/13 FOREIGN PATENTS 647,191 12/1950 Great Britain350/13 648,801 1/1951 Great Britain 350/13 FIG. la

k mm

VARIABLE PHASE CONTRAST MICROSCOPY The present invention relates toimprovements in microscopy with variable phase contrast observation; itlike wise relates to an optical device for observation by phase contrastof transparent or nonabsorbent objects or more generally objectsbelonging to the class of phase objects.

Numerous variable phase contrast devices have been proposed, all ofwhich contain polarizer means and a phase plate, but these devices havenot given the expected industry results. in these known devices forphase contrast observation, the basic phase shift, which must normallybe equal to irr/Z, is obtained by using an optical delay. In theseconditions, that is to say when an optical delay is brought into use,the phase p is expressed by: Fan/x) A, where the optical delay A is h isthe mean wavelength and it the wavelength conid red; he.resu thih t.Q(-EtrrlZ) o/? L which. P filH why in known devices, where an opticaldelay is used, the phase shift is equal to rr/Z only if l\0 and thebasic phase shift varies with the wavelength.

In order to avoid the disadvantages of known devices, which requiredouble regulation, it has been attempted to operate in accordance with aprocess-permitting regulation by a single control, so as to have thebenefit of the following advantages: firstly, this single control can becalibrated and thus supplies a means of measuring the optical thicknessof the object studied; on the other hand, by acting simultaneously onthe phase shift and on the absorption of the phase plate, said controlhas the effect that an object of any optical thickness, which is notnecessarily small in relation to the wavelength, appears with a contrastequal to I, that is to say the image of the detail studied becomes blackon a light background. This last phenomenon serves as a criterion ofperfect compensation and can therefore easily be used for measurements.

Starting from this basic idea of the present invention, the physical andmathematical theories established on phase contrat observation have beenreexamined in detail. lf the vectorial diagrams of phenomena areexamined with the object amplitude (Vector V [V =e "1), referring toFIG. 1 of the drawings and eliminating the arrows designating thevectors in order to simplify writing, it is seen that the vector V,'isbroken down into two vectors V and V in accordance with the vectorialequation V,=V +V FIG. la).

if it is desired to cancel the light inside the object, it is necessaryand sufficient that the vector V, of modulus equal to 1 should bereplaced by the vector V -V in this analysis the vector Vg, representsso-called direct light, which means that in order to extinguish theobject it is necessary to make a phase shift of V by the anglerl;=-(1r/2 p/2) and to reduce its modulus from 1 to 2 sin p/2. Thismeans that the optimum adjustment of the variable phase contrast isobtained by satisfying the following two simultaneous conditions:

1. The energy transparency of the so-called conjugate" zone of the phaseplate must be brought to the value 4 sin pl2=2 (l-cos p).

2. The phase shift it: of the said conjugate zone must be equal to(1r/2)+(p/2).

These considerations have been set out in detail by various authors(particularly on the one hand Bennett and others, Phase microscopy:Principles and applications" John Wiley & Sons, publishers, New York1957, and on the other hand Les contrastes de phase et le contraste parinterference (Conference of the lntemational Optics Commission, th-2l stMar. 1951), Maurice Francon, publishers, Editions de la Revue dOptique).

Various authors, for example, H. Osterberg with his POLANRET system andU.S. Pat. No. 2,516,905, have proposed the solution of the problem posedby forming the phase plate with two crossed polarizers followed by abirefringent quarter wave plate completed by a variable compensator, thewhole arrangement being disposed between a polarizer and an analyzer. Inthis case the quarter-wave plate effects the main phase shift equal to*'(1r/2), while the compensator makes it possible to add the small phaseshift equal to p/2. The transmission of the conjugate zone is thenadjusted by rotating the analyzer. It is seen immediately that thissolution, if used in the previously mentioned known devices, has the twodisadvantages already referred to, which the invention makes it possibleto avoid, as will be seen later on, and which are that:

l. optimum regulation must be effected by acting on two independentparameters, in order to adjust the latter;

2. the main phase shift of 1r/2 is obtained by means of a delay of M4which is essentially chromatic, because it depends on the wavelength ofthe illumination light.

Thus, going back to the conclusions drawn from the vectorial diagram inFIG. In, it was found that a solution of the problems posed could beobtained according to the present invention by treating the vector V,without subjecting it to the rotation, 11: and to the diminution ofmagnitude described in connection with FIG. In. If, as illustrated inFIG. lb, the vector (V,) is added to the vector V,,, there is obtained avector V defined by V V V, that is to say l-e This means that contrastcorresponding to the cancellation of light inside the object can beobtained by opposing the vector v, representing the coherent backgroundby a vector opposite to the vector V, representing the object. Theresult corresponding to ideal conditions of observation of an object ofphase p can be achieved by imparting, to the conjugate zone (representedby the vector V =le of the plate, the complex amplitude transmissionle"'and, to the complementary zone (vector V -l a transmission equal tol.

The improvements made in the observation variable phase contrastmicroscopy, utilizing a phase shift of rr/Z and a variable phase shift,consist fundamentally according to the invention in breaking down thephase plate into two zones known respectively as the conjugate and thecomplementary zone,

sisting of two polarizers turned by 45 in relation to one' another andrespectively occupying the conjugate zone and the complementary zone ofsaid plate, one polarizer being disposed in front of said plate in sucha manner that it covers the two aforesaid zones and that it is parallelto the polarizer occupying the conjugate zone of the plate, and anassembly composed in known manner of a birefringent compensator and ananalyzer being mounted behind the plate, in the direction of propagationof the light, in such a manner that the transmission of complexamplitudes of the conjugate zone will be equal to l-e and that of thecomplementary zone will be qsal l- The polarizers are advantageouslyjoined by adhesive bonding between two glass plates, the polarizersforming the phase plate having zones in which the polarizing action issuppressed by any suitable known photochemical treatment, said zonescovering respectively the conjugate zone and the complementary zone.

The compensator may may be of a SENARMONT type, which is known initself, and be provided with angular references making it possible tomeasure phase objects.

The phase plate is preferably placed in a plane conjugate to the focalplane of the objective in relation to an optical vehicle of amagnification preferably equal to -l.

The conjugate zone of the phase plate preferably has an annular zone therelative surface of which does not exceed 12 percent of the surfacecorresponding to the image of the outlet pupil of the objective used,which is projected on to the phase plate.

With reference to FIGS. 2 and 3 there are described below various formsof construction of an observation device according to the invention.

FIG. 1a and 1b represent phenomena occurring in known theoriesestablished on phase contrast observation.

FIG. 2 is a diagrammatic view of a first example of the invention,applied to microscopy.

FIG. 3 is a similar view of a modification.

FIGS. 4 and 5 show diagrams representative of phenomena occurring in theconjugate an the complementary zones.

FIGS. 6 and 7 are views, respectively in section and in plan, of thefirst polarizer and of the phase plate.

FIG. 8 is a diagram from which it is possible to determine the energytransmission T and the phase shift (41) of the conjugate zone of thephase plate in dependence on the phase p.

In the case of FIG. 2, a quasi-point source 1 transmits through acondenser 2 a parallel beam to the phase object (I. At the outlet of theobject O. the beam is broken down into a direct beam which, afterpassing through the objective 3, passes through a polarizer 4 focused onthe central portion of the phase plate 5, known as the conjugate zone,and a diffracted beam which passes entirely through the phase plate 5,being mainly affected by the transmission of the portion which surroundsthe conjugate zone and known as the complementary zone." The polarizer 4is of the undivided type. The phase plate 5 proper is constituted by apolarizer 6 having the same orientation as the polarizer 4 situated inthe conjugate zone, and a polarizer 7 oriented at 45 in relation to thepolarizer 4 and occupying the complementary zone (see FIGS. 6 and 7).

The phase plate 5, which in the example in placed in the focal plane Fof the objective 3, is conjugate to the source 1 in relation to theassembly comprising the condenser 2 and objective 3. After the plate 5the device comprises a birefringent compensator 8 symbolized by abirefringent plate of variable thickness and introducing a phasedifference I between the two waves polarized at 90 in relation to oneanother. The neutral lines of this compensator are directed at :45 inrelation to the polarizer 4. Finally, an analyzer 9 crossed with the.

polarizer 4 completes the arrangement. The real image of the object isprojected beyond the drawing and is not illustrated.

FIGS. 4 and 5 illustrate the evolution undergone respectively by thecomplex amplitudes of the direct wave and of the wave diffracted throughthe component elements of the device.

For the direct light, FIG. 4 the light polarized by the polarizer 4undergoes no modification through the effect of the polarizer 6, but itis decomposed by the compensator 8 into two perpendicular vibrations, ofwhich the two complex amplitudes are l/ and 1/ /5 The analyzer transmitsonly two antiparallel components of amplitudes k and a e For thediffracted light FIG. 5 it is seen that the polarizer 7 of thecomplementary zone transmits only a component directed at +45".

The compensator 8 does not modif its amplitude in anyway, the latterremaining equal to I finally, the analyzer 9 transmits the componentperpendicular to the polarizer 4 of amplitude A.

There is thus finally obtained a complex amplitude transmission ofdirect light equal to a constant close to le while that of the directlight is equal to 1.

By then bringing the phase shift I of the compensator 8 to the value pof the local object (I phase shift, an optimum image of the studieddetail of the object is obtained.

It is clearly seen that under the conditions of the invention the onlyvariable parameter is here the value 3 which is adjustable by means ofthe birefringent compensator 8 of known type.

Instead of the compensator illustrated in FIG. 2, it is possible to usewith advantage a SENARMONT compensator because of its simplicity andlinearity; as illustrated in FIG. 3, a

compensator of this type is composed of a quarter-wave plate It),usually of mica and the slow axis of which is parallel to the polarizer,and a rotating analyzer 9. A phase shift I is obtained by tumin theanalyzer through the angle 1 /2.

FIGS. 6 and show dismantled an advantageous form of construction of aphase plate composed of three polarizers 4, 6, and 7, assembled byadhesively bonding three polarizing sheets between two glass plates. Thefirst sheet 4 is in the undivided state, while the sheets 6 and 7 havenonpolarizing zones (6' and 7') which mutually complete one another. Theshape of the conjugate zone and also that of the source mayadvantageously be annular, as is well known.

It is not always materially possible to place the assembly constitutingthe phase plate 4, 6, 7 in the focal plane F of the objective 3. It isthen possible to use an optical vehicle of any type known in itselfwhich reproduces at a greater distance the real image of this focalplane, which is usually not accessible in a usual microscope objective.

In FIG. 8 there is illustrated a diagram permitting determination of thetransmission T in dependence on the phase p(cosinusoidal curve) and thephase shift I1! in dependence on the same phase p( broken line sawtoothcurve):

Iclaim:

I. In a method of performing variable phase contrast microscopicobservation, projecting light from a source toward an image through anoptical system having a phase plate and utilizing a main phase shift of1r/2 and a variable phase shift, the improvement comprising separatingthe phase plate into two zones, comprising a conjugate zone and acomplementary zone, and polarizing the light beam in two directions at45 to one another, one of the polarizations being operative in theconjugate zone and the other polarization being operative in thecomplementary zone, without optical path delay.

2. A device for variable phase contrast microscopic observation, a lightsource input comprising polarizing means, a phase plate, a birefringentcompensator, and an analyzer, and a light output, the phase plateconsisting of two polarizers operative at 45 to one another andrespectively occupying a conjugate zone and a complementary zone of saidplate, one polarizer being disposed in front of said plate and coveringthe two said zones and being parallel to the polarizer which occupiesthe conjugate zone of the plate, and an assembly composed of abirefringent compensator and an analyzer being mounted behind the plate,in the direction of propagation of the light, in a position compatiblewith the transmission of complex amplitudes of the conjugate zone beingequal to le and with that of the complementary zone being equal to l 3.A device as claimed in claim 2, wherein the polarizers are united byadhesive bonding between two glass plates and wherein the phase platethereby formed has zones in which polarizing action is suppressed, saidzone respectively covering the conjugate zone and the complementaryzone.

4. A device as claimed in claim 2, wherein the compensator is aSenannont compensator.

5. A device as claimed in claim 2, wherein said compensator is providedwith angular references enabling measurements of phase objects to beeffected.

6. A device as claimed in claim 2, wherein the phase plate is placed ina plane conjugate to the focal plane of the objective in relation to anoptical vehicle.

7. A device as claimed in claim 6, wherein said optical vehicle has amagnification equal to l.

8. A device as claimed in claim 2, wherein the conjugate zone of thephase plate has an annular zone the relative surface of which forms atmost 12 percent of the corresponding surface of the image of the outletpupil of the objective used, which is projected on to the phase plate.

=1: 1: s s a:

1. In a method of performing variable phase contrast microscopicobservation, projecting light from a source toward an image through anoptical system having a phase plate and utilizing a main phase shift ofpi /2 and a variable phase shift, the improvement comprising separatingthe phase plate into two zones, comprising a conjugate zone and acomplementary zone, and polarizing the light beam in two directions at45* to one another, one of the polarizations being operative in theconjugate zone and the other polarization being operative in thecomplementary zone, without optical path delay.
 2. A device for variablephase contrast microscopic observation, a light source input comprisingpolarizing means, a phase plate, a birefringent compensator, and ananalyzer, and a light output, the phase plate consisting of twopolarizers operative at 45* to one another and respectively occupying aconjugate zone and a complementary zone of said plate, one polarizerbeing disposed in front of said plate and covering the two said zonesand being parallel to the polarizer which occupies the conjugate zone ofthe plate, and an assembly composed of a birefringent compensator and ananalyzer being mounted behind the plate, in the direction of propagationof the light, in a position compatible with the transmission of complexamplitudes of the conjugate zone being equal to 1-ei and with that ofthe complementary zone being equal to
 1. 3. A device as claimed in claim2, wherein the polarizers are united by adhesive bonding between twoglass plates and wherein the phase plate thereby formed has zones inwhich polarizing action is suppressed, said zone respectively coveringthe conjugate zone and the complementary zone.
 4. A device as claimed inclaim 2, wherein the compensator is a Senarmont compensator.
 5. A deviceas claimed in claim 2, wherein said compensator is provided with angularreferences enabling measurements of phase objects to be effected.
 6. Adevice as claimed in claim 2, wherein the phase plate is placed in aplane conjugate to the focal plane of the objective in relation to anoptical vehicle.
 7. A device as claimed in claim 6, wherein said opticalvehicle has a magnification equal to -1.
 8. A device as claimed in claim2, wherein the conjugate zone of the phase plate has an annular zone therelative surface of which forms at most 12 percent of the correspondingsurface of the image of the outlet pupil of the objective used, which isprojected on to the phase plate.